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His tagging and 'cheaper' Nickel columns

Curt Ashendel ashendel at aclcb.purdue.edu
Fri Nov 17 11:20:20 EST 1995

On 17 Nov 1995 02:44:42 GMT, Dima Klenchin  <klenchin at macc.wisc.edu > 
>In article <50754.ashendel at aclcb.purdue.edu>,  "Curt Ashendel" wrote:

>>>Can someone recommend either an effective 'home-made' version of the Nickel 
>>>chelate methods (and a source for the materials), or give an assessment of the 
>>>relative merits of the above resins, and their re-useability?  
>>My comments are limited to home-made NTA-agarose:
>>I have successfully synthesized the NTA ligand and coupled it to 
>>agarose (Sepharose CL-4B). 

>[Synthesis of NTA deleted]

No, the synthesis described was not of NTA (nitrilotriaceetic acid), 
the diacetic acid derivative of glycine, but was 
N-(5-amino-1-carboxypentyl)iminodiacetic acid, the diacetic acid 
derivative of lysine.

>In fact Sigma sells very cheap NTA. 

Yes, but this NTA is the diacetic acid derivative of glycine, 
rather than lysine, and it has no functional group for linking to the 
sepharose.  This NTA sold by Signa is not useful for chromatography 
at all.

The reason I posted the comment about the synthesis of the lysine 
derivative is that this ligand (the lysine derivative) was not found 
to be for sale by any company, and must be synthesized.

>The real concern is activated Sepharose
>wich costs 10-fold over non-activated one. My choice would be epoxy
>activation but it generally results in slightly lower active groups density
>(I thinks). Should not matter, though (maybe nonspecific binding will be
>lower :-)) 

Actually, activated sepharoses are easy and inexpensive to make. The 
only problem that some people have with doing this themselves is that 
the reagents involved are very reactive (i.e., toxic/dangerous).

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu

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